Combination of 15N reverse labeling and afterglow spectroscopy for assigning membrane protein spectra by magic-angle-spinning solid-state NMR: application to the multidrug resistance protein EmrE

Magic-angle-spinning (MAS) solid-state NMR spectroscopy has emerged as a viable method to characterize membrane protein structure and dynamics. Nevertheless, the spectral resolution for uniformly labeled samples is often compromised by redundancy of the primary sequence and the presence of helical secondary structure that results in substantial resonance overlap. The ability to simplify the spectrum in order to obtain unambiguous site-specific assignments is a major bottleneck for structure determination. To address this problem, we used a combination of ¹⁵N reverse labeling, afterglow spectroscopic techniques, and frequency-selective dephasing experiments that dramatically improved the ability to resolve peaks in crowded spectra. This was demonstrated using the polytopic membrane protein EmrE, an efflux pump involved in multidrug resistance. Residues preceding the ¹⁵N reverse labeled amino acid were imaged using a 3D NCOCX afterglow experiment and those following were recorded using a frequency-selective dephasing experiment. Our approach reduced the spectral congestion and provided a sensitive way to obtain chemical shift assignments for a membrane protein where no high-resolution structure is available. This MAS methodology is widely applicable to the study of other polytopic membrane proteins in functional lipid bilayer environments.     

Substituted thieno[2,3-d]pyrimidines as adenosine A2A receptor antagonists

A novel series of benzyl substituted thieno[2,3-d]pyrimidines were identified as potent A_2A receptor antagonists. Several five- and six-membered heterocyclic replacements for the optimized methylfuran were explored. Select compounds effectively reverse catalepsy in mice when dosed orally.     

The LDL Receptor-Related Protein 1 (LRP1) Regulates the PDGF Signaling Pathway by Binding the Protein Phosphatase SHP-2 and Modulating SHP-2- Mediated PDGF Signaling Events

Background

The PDGF signaling pathway plays a major role in several biological systems, including vascular remodeling that occurs following percutaneous transluminal coronary angioplasty. Recent studies have shown that the LDL receptor-related protein 1 (LRP1) is a physiological regulator of the PDGF signaling pathway. The underlying mechanistic details of how this regulation occurs have yet to be resolved. Activation of the PDGF receptor β (PDGFRβ) leads to tyrosine phosphorylation of the LRP1 cytoplasmic domain within endosomes and generates an LRP1 molecule with increased affinity for adaptor proteins such as SHP-2 that are involved in signaling pathways. SHP-2 is a protein tyrosine phosphatase that positively regulates the PDGFRβ pathway, and is required for PDGF-mediated chemotaxis. We investigated the possibility that LRP1 may regulate the PDGFRβ signaling pathway by binding SHP-2 and competing with the PDGFRβ for this molecule.

Methodology/Principal Findings

To quantify the interaction between SHP-2 and phosphorylated forms of the LRP1 intracellular domain, we utilized an ELISA with purified recombinant proteins. These studies revealed high affinity binding of SHP-2 to phosphorylated forms of both LRP1 intracellular domain and the PDGFRβ kinase domain. By employing the well characterized dynamin inhibitor, dynasore, we established that PDGF-induced SHP-2 phosphorylation primarily occurs within endosomal compartments, the same compartments in which LRP1 is tyrosine phosphorylated by activated PDGFRβ. Immunofluorescence studies revealed colocalization of LRP1 and phospho-SHP-2 following PDGF stimulation of fibroblasts. To define the contribution of LRP1 to SHP-2-mediated PDGF chemotaxis, we employed fibroblasts expressing LRP1 and deficient in LRP1 and a specific SHP-2 inhibitor, NSC-87877. Our results reveal that LRP1 modulates SHP-2-mediated PDGF-mediated chemotaxis.

Conclusions/Significance

Our data demonstrate that phosphorylated forms of LRP1 and PDGFRβ compete for SHP-2 binding, and that expression of LRP1 attenuates SHP-2-mediated PDGF signaling events.     

One-carbon metabolism gene polymorphisms and risk of non-Hodgkin lymphoma in Australia

Dysregulation of the one-carbon metabolic pathway, which controls nucleotide synthesis and DNA methylation, may promote lymphomagenesis. We evaluated the association between polymorphisms in one-carbon metabolism genes and risk of non-Hodgkin lymphoma (NHL) in a population-based case-control study in Australia. Cases (n = 561) and controls (n = 506) were genotyped for 14 selected single-nucleotide polymorphisms in 10 genes (CBS, FPGS, FTHFD, MTHFR, MTHFS, MTR, SHMT1, SLC19A1, TCN1, and TYMS). We also conducted a meta-analysis of all studies of Caucasian populations investigating the association between MTHFR Ex5+79C > T (a.k.a., 677C>T) and NHL risk. A global test of 13 genotypes was statistically significant for diffuse large B-cell lymphoma (DLBCL; P = 0.008), but not for follicular lymphoma (FL; P = 0.27) or all NHL (P = 0.17). The T allele at MTHFR Ex5+79 was marginally significantly associated with all NHL (OR = 1.25, 95% CI = 0.98–1.59) and DLBCL (1.36, 0.96–1.93). The T allele at TYMS Ex8+157 was associated with a reduced risk of FL (0.64, 0.46–0.91). An elevated risk of NHL was also observed among carriers of the G allele at FTHFD Ex21+31 (all NHL, 1.31, 1.02–1.69; DLBCL, 1.50, 1.05–2.14). A meta-analysis of 11 studies conducted in Caucasian populations of European origin (4,121 cases and 5,358 controls) supported an association between the MTHFR Ex5+79 T allele and increased NHL risk (additive model, P = 0.01). In conclusion, the results of this study suggest that genetic polymorphisms of one-carbon metabolism genes such as MTHFR and TYMS may influence susceptibility to NHL. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Duplication and selection on abalone sperm lysin in an allopatric population

While gene duplication is a major source of evolutionary novelty, the importance of this process in reproductive protein evolution has not been widely investigated. Here, we report the first known case of gene duplication of abalone sperm lysin in an allopatric subspecies found in the Eastern Atlantic, Haliotis tuberculata coccinea. Mass spectrometry identified both copies of the lysin protein in testis tissue, and 3-dimensional structural modeling suggests that both proteins remain functional. We also detected positive selection acting on both paralogs after duplication and found evidence of a recent selective sweep. Because H. t. coccinea occurs in geographic isolation from other abalone species, these findings suggest that the evolution of lysin is not driven to create reproductive barriers to unfit hybrid formation with an overlapping species. Instead, sexual selection or sexual conflict acting during abalone fertilization could be responsible for the recent positive selection on this protein. The presence of multiple, rapidly evolving lysin genes in H. tuberculata presents an opportunity to study the early stages of diversification of a protein whose function is well understood.     

Comparative morphology of freshwater sculpin inhabiting different environmental conditions in the Chesapeake Bay headwaters

Environmental Biology of Fishes - We compared body morphology of two freshwater sculpin taxa that inhabit distinct environmental conditions in the Chesapeake Bay watershed of eastern North America:...     

Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40^phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1''s function in endocytosis.     

DNAH5 is associated with total lung capacity in chronic obstructive pulmonary disease

Background

Chronic obstructive pulmonary disease (COPD) is characterized by expiratory flow limitation, causing air trapping and lung hyperinflation. Hyperinflation leads to reduced exercise tolerance and poor quality of life in COPD patients. Total lung capacity (TLC) is an indicator of hyperinflation particularly in subjects with moderate-to-severe airflow obstruction. The aim of our study was to identify genetic variants associated with TLC in COPD.

Methods

We performed genome-wide association studies (GWASs) in white subjects from three cohorts: the COPDGene Study; the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE); and GenKOLS (Bergen, Norway). All subjects were current or ex-smokers with at least moderate airflow obstruction, defined by a ratio of forced expiratory volume in 1 second to forced vital capacity (FEV₁/FVC) <0.7 and FEV₁ < 80% predicted on post-bronchodilator spirometry. TLC was calculated by using volumetric computed tomography scans at full inspiration (TLC_CT). Genotyping in each cohort was completed, with statistical imputation of additional markers. To find genetic variants associated with TLC_CT, linear regression models were used, with adjustment for age, sex, pack-years of smoking, height, and principal components for genetic ancestry. Results were summarized using fixed-effect meta-analysis.

Results

Analysis of a total of 4,543 COPD subjects identified one genome-wide significant locus on chromosome 5p15.2 (rs114929486, β = 0.42L, P = 4.66 × 10⁻⁸).

Conclusions

In COPD, TLC_CT was associated with a SNP in dynein, axonemal, heavy chain 5 (DNAH5), a gene in which genetic variants can cause primary ciliary dyskinesia. DNAH5 could have an effect on hyperinflation in COPD.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-014-0097-y) contains supplementary material, which is available to authorized users.